The so-called tRNAGlu1 of Escherichia coli is a stable denatured conformer of the major isoacceptor tRNAGlu2

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.     

A single glutamyl-tRNA synthetase aminoacylates tRNAGlu and tRNAGln in Bacillus subtilis and efficiently misacylates Escherichia coli tRNAGln1 in vitro.

In the presence or absence of its regulatory factor, the monomeric glutamyl-tRNA synthetase from Bacillus subtilis can aminoacylate in vitro with glutamate both tRNAGlu and tRNAGln from B. subtilis and tRNAGln1 but not tRNAGln2 or tRNAGlu from Escherichia coli. The Km and Vmax values of the enzyme for its substrates in these homologous or heterologous aminoacylation reactions are very similar. This enzyme is the only aminoacyl-tRNA synthetase reported to aminoacylate with normal kinetic parameters two tRNA species coding for different amino acids and to misacylate at a high rate a heterologous tRNA under normal aminoacylation conditions. The exceptional lack of specificity of this enzyme for its tRNAGlu and tRNAGln substrates, together with structural and catalytic peculiarities shared with the E. coli glutamyl- and glutaminyl-tRNA synthetases, suggests the existence of a close evolutionary linkage between the aminoacyl-tRNA synthetases specific for glutamate and those specific for glutamine. A comparison of the primary structures of the three tRNAs efficiently charged by the B. subtilis glutamyl-tRNA synthetase with those of E. coli tRNAGlu and tRNAGln2 suggests that this enzyme interacts with the G64-C50 or G64-U50 in the T psi stem of its tRNA substrates.     

Formation of the chlorophyll precursor delta-aminolevulinic acid in cyanobacteria requires aminoacylation of a tRNAGlu species.

In the chloroplasts of higher plants and algae, the biosynthesis of the chlorophyll precursor delta-aminolevulinic acid (ALA) involves at least three enzymes and a tRNA species. Here we demonstrate that in cell extracts of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 ALA was formed from glutamate in a series of reactions in which activation of glutamate by glutamyl-tRNAGlu formation was the first step. The activated glutamate was reduced by a dehydrogenase which displayed tRNA sequence specificity. Fractionation of strain 6803 tRNA by reverse-phase chromatography and polyacrylamide gel electrophoresis yielded two pure tRNAGlu species which stimulated ALA synthesis in vitro. These tRNAs had identical primary sequences but differed in the nucleotide modification of their anticodon. The 6803 tRNAGlu was similar to the sequences of tRNAGlu species or tRNAGlu genes from Escherichia coli and from chloroplasts of Euglena gracilis and higher plants. Southern blot analysis revealed at least two tRNAGlu gene copies in the 6803 chromosome. A glutamate-1-semialdehyde aminotransferase, the terminal enzyme in the conversion of glutamate to ALA in chloroplasts, was detected in 6803 cell extracts by the conversion of glutamate-1-semialdehyde to ALA and by the inhibition of this reaction by gabaculin.     

Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity

In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.     

Effect of modified nucleotides on Escherichia coli tRNAGlu structure and on its aminoacylation by glutamyl-tRNA synthetase. Predominant and distinct roles of the mnm5 and s2 modifications of U34.

Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP.     

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.     

Specific incorporation of selenium into lysine- and glutamate- accepting tRNAs from Escherichia coli

Amino acid transfer nucleic acids (tRNAs) that contain selenium-modified bases are synthesized by Escherichia coli in the presence of low levels (0.1-0.5 microM) of [75Se]selenite or [75Se]selenate. The amount of selenium incorporated (1-2 g atoms/100 mol of tRNA) was unchanged by 10-20-fold variations in selenium or sulfate concentrations or by the addition of 1 mM cysteine, sulfide, or sulfite. Specific incorporation of selenium (as opposed to nonspecific substitution for sulfur) was further indicated by the different reversed phase chromatographic elution patterns of 35S- and 75Se-labeled tRNAs isolated from cells labeled with 35SO2-4 and 75SeO2-4. Also, E. coli mutants unable to synthesize an abundant sulfur-modified base, 4-thiouracil, nevertheless produced normal levels of selenium-modified tRNAs. Two different methods of distinguishing between aminoacylated and nonaminoacylated tRNA, one which examined mobility during reversed phase chromatography and another which employed anti-AMP antibodies, indicated that over 50% of the selenium-containing tRNA had lysine or glutamate acceptor activity.     

Ty1 and delta elements occur adjacent to several tRNA genes in yeast

A comparative analysis of a number of yeast DNA-pBR322 recombinant plasmids carrying repetitive sequence elements has revealed that Ty1 or delta elements occur in the vicinity of several tRNA genes. Four examples have been characterized in detail: three glutamate tRNA genes and a serine tRNA gene. The tRNAGlu3 genes occupy different chromosomal locations; two of these genes are found adjacent to Ty1 elements, and the third is found adjacent to an independent delta element. A delta unit is also found adjacent to a tRNASer2 gene. Next to one of the tRNAGlu3 genes, the delta element is joined to a truncated sigma element. Junctions between different delta units were characterized by the sequence analysis of two DNA segments that carry no tRNA genes.     

Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. Study of the interactions with its substrates.

The binding of the various substrates to Escherichia coli glutamyl-tRNA synthetase has been investigated by using as experimental approaches the binding study under equilibrium conditions and the substrate-induced protection of the enzyme against its thermal inactivation. The results show that ATP and tRNAGlu bind to the free enzyme, whereas glutamate binds only to an enzyme form to which glutamate-accepting tRNAGlu is associated. By use of modified E. coli tRNAsGlu and heterologous tRNAsGlu, a correlation could be established between the ability of tRNAGlu to be aminoacylated by glutamyl-tRNA synthetase and its abilities to promote the [32P]PPi-ATP isotope exchange and the binding of glutamate to the synthetase. These results give a possible explanation for the inability of blutamyl-tRNA synthetase to catalyze the isotope exchange in the absence of amino acid accepting tRNAGlu and for the failure to detect an enzyme-adenylate complex for this synthetase by using the usual approaches. One binding site was detected for each substrate. The specificity of the interaction of the various substrates has been further investigated. Concerning ATP, inhibition studies of the aminoacylation reaction by various analogues showed the existence of a synergistic effect between the adenine and the ribose residues for the interaction of adenosine. The primary recognition of ATP involves the N-1 and the 6-amino group of adenine as well as the 2'-OH group of ribose. This first interaction is then strengthened by the phosphate groups- Inhibition studies by various analogues of glutamate showed a strong decrease in the affinity of this substrate for the synthetase after substitution of the alpha- or gamma-carboxyl groups. The enzyme exhibits a marked tendency to complex tRNAs of other specificities even in the presence of tRNAGlu. MgCl2 and spermidine favor the specific interactions. The influence of monovalent ions and of pH on the interaction between glutamyl-tRNA synthetase and tRNAGlu is similar to those reported for other synthetases not requiring their cognate tRNA to bind the amino acid. Finally, contrary to that reported for other monomeric synthetases, no dimerization of glutamyl-tRNA synthetase occurs during the catalytic process.     

Isolation and partial characterization of selenium-containing tRNA from germinating barley

Selenium-containing tRNA was discovered in germinating barley for the first time with the ⁷⁵Se isotopic tracer technique; therefore, this technique was used to study the effect of different concentrations of selenium and sulfur in the medium on the content of selenium-containing tRNA in germinating barley. Se-containing tRNAs and its hydrolysates were isolated, purified, and characterized by means of column chromatography, ion-exchange chromatography, high-performance liquid chromatography, and the ultraviolet-visible spectrum. The results show that the amount of selenium in tRNA is almost unaffected by the sulfuric content in the medium, and the pathway for selenium and sulfur to enter tRNA might not be exactly the same. Selenium exists within tRNA in the form of 5-methylamine methyl-2-selenouridine, just as it does within a microorganism tRNA.     

The nucleotide sequence of a glutamine tRNA from rat liver.

A glutamate tRNA from rat liver was purified. By means of post-labeling techniques, its nucleotide sequence was shown to be: pU-C-C-C-A-C-A-U-m1G-G-U-C-psi-A-G-C- G-G-D-D-A-G-G-A-U-U-C-C-U-G-G-psi-U-mcm5S2U-U-C-A-C-C-C-A-G-G-C-G- G-C-m5C-m5C-G-G-G-Tm-psi-C-G-A-C-U-C-C-C-G-G-U-G-U-G-G-G-A-A-C-C-AOH. The sequence is remarkably similar to that of tRNAGlu from Drosophila melanogaster. Only 10 out of 75 nucleotides in the two tRNAs are different.     

Specific spin-labeling of transfer ribonucleic acid molecules.

The spin labels anhydride (ASL), bromoacetamide (BSL) and carbodiimide (CSL) were used to label selectively tRNAGlu, tRNA fMet and tRNAPhe from E. coli. The preparation and characterization of the sites of labeling of eight new spin-labeled tRNAs are described. The sites of labeling are: s2U using ASL, BSL and CLS and tRNAGlu; s4U using ASL and BSL on tRNAfMet and tRNAPhe; U-37 with CSL on tRNfMet; U-33 with CSL on tRNAPhe. The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.     

Sequence analysis of the glutamate tRNA family: evidence for pseudogenes

Human tRNA(CUCGlu) has been isolated by direct hybridization of the tRNA to 28S ribosomal RNA. We now report the isolation of mouse tRNA(CUCGlu) using the same procedure. Partial sequence analysis of the mouse tRNA shows that it is identical to the human tRNA and to a cloned rat tDNA(CUCGlu) sequence. This mouse tRNA(CUCGlu), however, differs by one nucleotide from a previously cloned mouse tDNA(CUCGlu) sequence, suggesting that the tDNA may be a pseudogene. Further evolutionary comparison of these and other glutamate tRNAs and tDNAs has provided evidence to suggest that two other tDNA(Glu) sequences arose by mutation of functional tRNAGlu genes such that their anticodon sequences were converted from one glutamate isoacceptor to the other. These tDNA sequences may also represent pseudogenes.     

Editing corrects mispairing in the acceptor stem of bean and potato mitochondrial phenylalanine transfer RNAs.

Editing is a general event in plant mitochondrial messenger RNAs, but has never been detected in a plant mitochondrial transfer RNA (tRNA). We demonstrate here the occurrence of a tRNA editing event in higher plant mitochondria: in both bean and potato, the C encoded at position 4 in the mitochondrial tRNA(Phe)(GAA) gene is converted into a U in the mature tRNA. This nucleotide change corrects the mismatched C4-A69 base-pair which appears when folding the gene sequence into the cloverleaf structure and it is consistent with the fact that C to U transitions constitute the common editing events affecting plant mitochondrial messenger RNAs. The tRNA(Phe)(GAA) gene is located upstream of the single copy tRNA(Pro)(UGG) gene in both the potato and the bean mitochondrial DNAs. The sequences of potato and bean tRNA(Pro)(UGG) genes are colinear with the sequence of the mature bean mitochondrial tRNA(Pro)(UGG), demonstrating that this tRNA is not edited. A single copy tRNA(Ser)(GCU) gene was found upstream of the tRNA(Phe) gene in the potato mitochondrial DNA. A U6-U67 mismatched base-pair appears in the cloverleaf folding of this gene and is maintained in the mature potato mitochondrial tRNA(Ser)(GCU), which argues in favour of the hypothesis that the editing system of plant mitochondria can only perform C to U or occasionally U to C changes.     

Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli tRNA

A selenium-containing nucleoside, 5-methylaminomethyl-2-selenouridine (mnm5se2U), is present in lysine- and glutamate-isoaccepting tRNA species of Escherichia coli. The synthesis of mnm5se2U is optimum (4 mol/100 mol tRNA) when selenium is present at about 1 microM concentration and is neither decreased by a high (8 mM) level of sulfur in the medium nor increased by excessive (10 or 100 microM) levels of selenium. Lysine- and glutamate-isoaccepting tRNA species that contain 5-methylaminomethyl-2-thiouridine (mnm5s2U) coexist with the seleno-tRNAs in E. coli cells and a reciprocal relationship between the mnm5se2U- and the mnm5s2U-containing species is maintained under a variety of growth conditions. The complete 5-methylaminomethyl side chain is not a prerequisite for introduction of selenium at the 2-position. In E. coli mutants deficient in the ability to synthesize the 5-methylaminomethyl substituent, both the 2-thiouridine and the corresponding 2-selenouridine derivatives of intermediate forms are accumulated. Broken cell preparations of E. coli synthesize mnm5se2U in tRNAs by an ATP-dependent process that appears to involve the replacement of sulfur in mnm5s2U with selenium.     

Sequence analysis of two yeast mitochondrial DNA fragments containing the genes for tRNA Ser UCR and tRNA Phe UUY.

Two restriction enzyme fragments containing yeast mitochondrial tRNA genes have been characterized by DNA sequence analysis. One of these fragments is 320 base pairs long and contains a tRNA Ser gene. The corresponding tRNA SER was isolated from yeast mitochondria and its nucleotide sequence also was determined. This mitochondrial tRNA is 90 nucleotides in length, has a G + C content of 38%, and has UGA as the anticodon. A portion of a 680-base-pair DNA fragment containing a tRNA Phe gene was also sequenced. The portion of this gene which codes for the mature tRNA is 75 base pairs in length, has a G + C content of 33%, and contains the anticodon GAA. Neither gene contains an intervening sequence or codes for the 3' CCA terminus. Both are surrounded by regions of more than 90% A + T. The significance of these sequences is discussed.     

Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions.